Plasmids as Vectors of Antibiotic Resistance: Evolution of Plasmid Host Range The dramatic spread of antibiotic resistance is a crisis in the treatment of infectious diseases that affect humans. Although plasmid-mediated gene transfer is now recognized as an important means for the spread of drug resistance, very little is known about the range of hosts to which plasmids can transfer, or if this range evolves over time. While some plasmids only transfer and stably replicate in a narrow range of hosts, so-called broad-host-range plasmids can transfer and replicate in distantly related bacteria, thereby shuffling resistance genes across taxonomic barriers. Understanding the features of the broad-host-range plasmids responsible for their ability to function in a wide range of bacterial hosts is therefore of both medical and fundamental interest. The research proposed builds on and extends the findings of previous studies, which showed that various mutations in the plasmid encoded Rep protein (TrfA) required for plasmid replication are sufficient to alter the host range of a broad-host-range (BHR) plasmid. The long-term goal of this project is to gain insight into the evolutionary patterns of host range shifts of drug resistance plasmids. Here we will explore the general mechanisms and dynamics of plasmid host range evolution by integrating data from experimental evolution of BHR and narrow host range (NHR) plasmids with statistical modeling of plasmid evolutionary dynamics. In this multi-disciplinary study, we propose to address the following specific aims: (1) Determine the evolutionary mechanisms of host range shifts by BHR and NHR plasmids;(2) Develop statistical models and simulations of plasmid evolutionary dynamics;(3) Determine the molecular mechanisms of plasmid host range shifts. In the future, this fundamental knowledge will support research into drug therapies based on restricting the horizontal transfer or stable replication of drug resistance or virulence plasmids in human pathogens. Restricting the spread and persistence of unwanted plasmids is a novel and promising avenue in the fight against human pathogens that could very well be part of future strategies to avoid escalation of this health crisis. PUBLIC HEALTH RELEVANCE: This work will provide a roadmap for further mechanistic studies on the role of specific mutations in plasmid host-range and may lead to attractive therapies that target the replication of antibiotic resistance plasmids and limit the spread of antibiotic resistance in human pathogens.